Chemo-/Regio-Selective Synthesis of Novel Functionalized Spiro[pyrrolidine-2,3′-oxindoles] under Microwave Irradiation and Their Anticancer Activity

A novel series of nitrostyrene-based spirooxindoles were synthesized via the reaction of substituted isatins 1a–b, a number of α-amino acids 2a–e and (E)-2-aryl-1-nitroethenes 3a–e in a chemo/regio-selective manner using [3+2] cycloaddition (Huisgen) reaction under microwave irradiation conditions. The structure elucidation of all the synthesized spirooxindoles were done using 1H and 13C NMR and HRMS spectral analysis. The single crystal X-ray crystallographic study of compound 4l was used to assign the stereochemical arrangements of the groups around the pyrrolidine ring in spiro[pyrrolidine-2,3′-oxindoles] skeleton. The in vitro anticancer activity of spiro[pyrrolidine-2,3′-oxindoles] analogs 4a–w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines shows promising results. Out of the 23 synthesized spiro[pyrrolidine-2,3′-oxindoles], while five compounds (4c, 4f, 4m, 4q, 4t) (IC50 = 34.99–47.92 µM; SI = 0.96–2.43) displayed significant in vitro anticancer activity against human lung (A549) cancer cell lines, six compounds (4c, 4f, 4k, 4m, 4q, 4t) (IC50 = 41.56–86.53 µM; SI = 0.49–0.99) displayed promising in vitro anticancer activity against human liver (HepG2) cancer cell lines. In the case of lung (A549) cancer cell lines, these compounds were recognized to be more efficient and selective than standard reference artemisinin (IC50 = 100 µM) and chloroquine (IC50 = 100 µM; SI: 0.03). However, none of them were found to be active as compared to artesunic acid [IC50 = 9.85 µM; SI = 0.76 against lung (A549) cancer cell line and IC50 = 4.09 µM; SI = 2.01 against liver (HepG2) cancer cell line].


Introduction
In the search for a tool to synthesize a variety of pharmaceutically important heterocycles, one methodology that conceivably meets all the goals and gives a better alternative for library creation of heterocycles is the "multicomponent reaction" pathway.Moreover, it introduces the complexity, regiospecificity and stereoselectivity in the minimum number of steps [1][2][3].The combination of all these properties along with the very quick reaction kinetics of microwave contributes new strategies to the fast and productive combination of heterocycles.Since the development of precisely regulated microwave reactors, microwaveassisted organic synthesis (MAOS) has efficiently revolutionized synthetic heterocyclic chemistry.The microwave reactor has been utilized to quicken the reaction rate along with the enhancement in the yields of the desired product, shortening the reaction time and upgrading the selectiveness of the reaction [4][5][6].In this sequence, microwave-assisted synthesis of a series of distinctive nitrostyrene-based spirooxindoles as a potent anticancer agent has been done against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines.
Cancer is the primary cause of death in both more and less developed countries and the burden is anticipated to increase globally owing to population growth and aging.According to estimates, approximately 19.3 million new cancer diagnoses and 10.0 million casualties happened globally in 2020 based on GLOBACON estimates [7].Cancer is an abnormal development of cells that, in general, multiply in an uncontrolled manner.For several years, lung cancer has been the most frequently diagnosed and the prime cause of death in men and the second major cause of casualties in women [8].Liver cancer is the sixth most common cancer globally.In addition, previous studies from the year 2020 depicted that approx.905,677 people were diagnosed with liver cancer worldwide.Out of these, 830,200 people died from liver cancer throughout the world.By 2040, there may be a >55% increase in the number of new liver cancer cases and fatalities [9].Numerous natural and synthetic anticancer drugs are accessible in the market (cisplatin and paclitaxel); nevertheless, such pharmaceutical products are often associated with unfortunate and undesirable side effects [10].Hence, the significance of investigating new prominent pharmaceutically important skeletons as anticancer agents through easily accessible starting materials becomes a crucial step in improving the success rates in cancer treatment.
Spirooxindoles, specifically the spiro(pyrrolidine-2,3 -oxindole) core as a ubiquitous pharmaceutically privileged heterocyclic moiety, are blended with numerous significant biological activities (Figure 1) such as anticancer [11][12][13][14][15], antimicrobial [16][17][18][19], anti-inflammatory [20,21], antimycobacterial [22], acetylcholinesterase-inhibitory [23,24], MDM2-p53 interaction inhibitor [25], p38alpha inhibitor [26], β-secretase (BACE1) inhibitory activity [27], antitumor [28], antitubercular [29], antimalarial [30], etc.These spiro molecules are the key skeleton of many bioactive natural/synthetic scaffolds such as horsfiline [31][32][33][34][35], MI-219 [36], rhynchophylline and isorhynchophylline [37], mitraphylline [38], elacomine [39], alstonine [40], spiro-tryprostatine A and B [41,42], etc.In view of these facts, spirooxindoles can be described as being on the verge of becoming a new anticancer lead molecule because similar skeleton-based lead molecules such as MI-888 and NITD609 are now in preclinical assessments for the treatment of malaria and cancer growth individually [43,44].Therefore, inspired by MI-888 and MI-219, which contain an oxindole substructure, i.e.; spiro(pyrrolidine-2,3 -oxindole) motifs with a spiro pyrrolidine ring having nitrogen at N3 position and as inhibitors of MDM2-p53 are potential anti-breast cancer agents; herein, we present the design, and synthesize and evaluate the in vitro anticancer activity of a novel series of nitrostyrene-based spirooxindoles in which the oxindole core with spiro pyrrolidine ring has nitrogen in the N2 location, i.e., spiro(pyrrolidine-2,3 -oxindole) as the key pharmacophore.Nitroaromatic substructure has been identified in several literature studies as a potentially effective cytotoxic agent, either in part or as a whole [44][45][46][47].The reduction of the nitro group, subsequently followed by the interaction of the active intermediate species (nitro-radical anions) with DNA, is the primary cause of the biological activity of nitro derivatives [48].Various reports depict that the introduction of a nitro group on bioactive moieties enhances their biological activity [49][50][51][52][53].However, in this sequence, inspired by the naturally and synthetically occurring cytotoxic and anticancer pharmacophore, i.e.; Wuweizi B [54][55][56], Buflavine [57][58][59] and nitrovinylstilbenes [60][61][62], respectively, we envisaged that the hybrid of donating group substituting nitroaromatic system with spirooxindole core moiety could lead to a further increase in cytotoxicity and, thus, increase the anticancer activity.The purpose of taking the bromo derivative of isatin can be justified by the fact that it carries a variety of biological activities including anticancer ones and the spiro derivatives of bromoisatin also have antiphlogistic and analgesic activities.Moreover, taking into account the cytotoxic activity of naturally occurring tyrindoleninone, we decided to add such a kind of bromo derivative of isatin into our design methodology [63][64][65][66][67]. Therefore, considering all the facts, we designed a hybrid prototype (Figure 2).Although there are only a few reports available in the literature on nitrostyrene-based spirooxindoles [68][69][70][71][72], and several types of spirooxindoles have also been highlighted, only a few of them emphasized their anticancer activity [73][74][75][76][77][78][79].Herein, in pursuit of a new anticancer agent using our designed prototype, for the first time we report the synthesis and in vitro anticancer activities of twenty-three various nitrostyrene-based spirooxindole derivatives 4a-w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines.The anticancer drugs artemisinin, chloroquine, artesunic acid and Paclitaxel were taken as standard reference.The stereochemistry of the target compound was confirmed by X-ray crystal analysis (Scheme 1).Nitroaromatic substructure has been identified in several literature studies as a potentially effective cytotoxic agent, either in part or as a whole [44][45][46][47].The reduction of the nitro group, subsequently followed by the interaction of the active intermediate species (nitro-radical anions) with DNA, is the primary cause of the biological activity of nitro derivatives [48].Various reports depict that the introduction of a nitro group on bioactive moieties enhances their biological activity [49][50][51][52][53].However, in this sequence, inspired by the naturally and synthetically occurring cytotoxic and anticancer pharmacophore, i.e.; Wuweizi B [54][55][56], Buflavine [57][58][59] and nitrovinylstilbenes [60][61][62], respectively, we envisaged that the hybrid of donating group substituting nitroaromatic system with spirooxindole core moiety could lead to a further increase in cytotoxicity and, thus, increase the anticancer activity.The purpose of taking the bromo derivative of isatin can be justified by the fact that it carries a variety of biological activities including anticancer ones and the spiro derivatives of bromoisatin also have antiphlogistic and analgesic activities.Moreover, taking into account the cytotoxic activity of naturally occurring tyrindoleninone, we decided to add such a kind of bromo derivative of isatin into our design methodology [63][64][65][66][67]. Therefore, considering all the facts, we designed a hybrid prototype (Figure 2).Although there are only a few reports available in the literature on nitrostyrene-based spirooxindoles [68][69][70][71][72], and several types of spirooxindoles have also been highlighted, only a few of them emphasized their anticancer activity [73][74][75][76][77][78][79].Herein, in pursuit of a new anticancer agent using our designed prototype, for the first time we report the synthesis and in vitro anticancer activities of twenty-three various nitrostyrene-based spirooxindole derivatives 4a-w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines.The anticancer drugs artemisinin, chloroquine, artesunic acid and Paclitaxel were taken as standard reference.The stereochemistry of the target compound was confirmed by X-ray crystal analysis (Scheme 1).Scheme 1.Our approach: Synthesis of nitrostyrene-based spirooxindole derivatives 4a-w.

Synthesis
Our initial investigation was aimed towards the development of a sustainable and an environmentally benign economic protocol for the synthesis of spiro[pyrrolidine-2,3′-oxindoles].We started our optimization study by utilizing the multicomponent reaction of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a as a starting substrate (Table 1) under conventional heating as well as under microwave irradiation conditions.The study involving conventional heating furnished the desired 4b from the reaction of 1a, 2b and 3a in up to a maximum 65% yield at r.t to 100 °C temperature range and in 120-240 min time range (Table 1, entry 1-14).Then, we switched our attention towards microwave-assisted optimization study for the synthesis of spiro[pyrrolidine-2,3′-oxin- Scheme 1.Our approach: Synthesis of nitrostyrene-based spirooxindole derivatives 4a-w.

Synthesis
Our initial investigation was aimed towards the development of a sustainable and an environmentally benign economic protocol for the synthesis of spiro[pyrrolidine-2,3′-oxindoles].We started our optimization study by utilizing the multicomponent reaction of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a as a starting substrate (Table 1) under conventional heating as well as under microwave irradiation conditions.The study involving conventional heating furnished the desired 4b from the reaction of 1a, 2b and 3a in up to a maximum 65% yield at r.t to 100 °C temperature range and in 120-240 min time range (Table 1, entry 1-14).Then, we switched our attention towards microwave-assisted optimization study for the synthesis of spiro[pyrrolidine-2,3′-oxin-Scheme 1.Our approach: Synthesis of nitrostyrene-based spirooxindole derivatives 4a-w.

Synthesis
Our initial investigation was aimed towards the development of a sustainable and an environmentally benign economic protocol for the synthesis of spiro[pyrrolidine-2,3oxindoles].We started our optimization study by utilizing the multicomponent reaction of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a as a starting substrate (Table 1) under conventional heating as well as under microwave irradiation conditions.The study involving conventional heating furnished the desired 4b from the reaction of 1a, 2b and 3a in up to a maximum 65% yield at r.t to 100 • C temperature range and in 120-240 min time range (Table 1, entry 1-14).Then, we switched our attention towards microwave-assisted optimization study for the synthesis of spiro[pyrrolidine-2,3 -oxindoles].Hitherto, it is widely mentioned that water can be an excellent solvent for the synthesis of spirooxindole [80][81][82].Therefore, we started our investigation by the reaction of an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a in aqueous medium at room temperature for 30 min under microwave irradiation.Unfortunately, 4b was obtained only in a trace amount (Table 1, entry 1), whereas reducing the time of microwave to 20 and 10 min does not generate the product 4b at all (Table 1, entries 2-3).Furthermore, it is also reported that polar solvents such as EtOH, MeOH and/or their aqueous mixture have been used for the synthesis of spirooxindole [11][12][13][14][15]. Consequently, we decided to take an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a dissolved in equal ratio of MeOH: H 2 O (1:1) at a temperature of 60 • C, 90 • C and 120 • C for 15, 10 and 5 min under microwave irradiation, respectively.However, intriguingly, it furnished 4b in 66%, 62% and 48% yields, respectively (Table 1, entries [4][5][6].Keeping all the other reaction conditions the same, when EtOH was used instead of MeOH along with an equal ratio of water at a temperature of 60 • C, 90 • C and 120 • C for 15, 10 and 5 min under microwave irradiations, respectively, 4b was obtained in 59%, 49% and 36% yields, respectively (Table 1, entries 10-12).It was confirmed that when using water, either alone or in a mixture with polar solvents, the reaction does not give fruitful results.Thus, we decided to use only polar solvents in our further optimization studies.In the quest for an effectual result, keeping all the other reaction conditions the same at a temperature of 60 • C for 15, 10 and 5 min, respectively, 4b was obtained in 91%, 82% and 61% yields, respectively (Table 1, entries 7-9).Subsequently, keeping all the reaction variables the same, the effect of variation in the temperature was examined.It was observed that increasing the temperature up to 90 • C and 120 • C does not have any beneficial effect on the yield of the reaction (Table 1, entries [13][14].Thus, overall, an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4dimethoxy-2-(2-nitrovinyl)benzene 3a dissolved in methanol as solvent at 60 • C for 15 min under microwave irradiation was found to be the best optimized reaction condition.doles].Hitherto, it is widely mentioned that water can be an excellent solvent for the synthesis of spirooxindole [80][81][82].Therefore, we started our investigation by the reaction of an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a in aqueous medium at room temperature for 30 min under microwave irradiation.Unfortunately, 4b was obtained only in a trace amount (Table 1, entry 1), whereas reducing the time of microwave to 20 and 10 min does not generate the product 4b at all (Table 1, entries 2-3).Furthermore, it is also reported that polar solvents such as EtOH, MeOH and/or their aqueous mixture have been used for the synthesis of spirooxindole [11][12][13][14][15]. Consequently, we decided to take an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a dissolved in equal ratio of MeOH: H2O (1:1) at a temperature of 60 °C, 90 °C and 120 °C for 15, 10 and 5 min under microwave irradiation, respectively.However, intriguingly, it furnished 4b in 66%, 62% and 48% yields, respectively (Table 1, entries [4][5][6].Keeping all the other reaction conditions the same, when EtOH was used instead of MeOH along with an equal ratio of water at a temperature of 60 °C, 90 °C and 120 °C for 15, 10 and 5 min under microwave irradiations, respectively, 4b was obtained in 59%, 49% and 36% yields, respectively (Table 1, entries 10-12).It was confirmed that when using water, either alone or in a mixture with polar solvents, the reaction does not give fruitful results.Thus, we decided to use only polar solvents in our further optimization studies.In the quest for an effectual result, keeping all the other reaction conditions the same at a temperature of 60 °C for 15, 10 and 5 min, respectively, 4b was obtained in 91%, 82% and 61% yields, respectively (Table 1, entries 7-9).Subsequently, keeping all the reaction variables the same, the effect of variation in the temperature was examined.It was observed that increasing the temperature up to 90 °C and 120 °C does not have any beneficial effect on the yield of the reaction (Table 1, entries 13-14).Thus, overall, an equimolar amount of isatin 1a, L-alanine 2b and (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a dissolved in methanol as solvent at 60 °C for 15 min under microwave irradiation was found to be the best optimized reaction condition.With the optimized reaction conditions in hand, we next explored the substrate scope utilizing substituted isatins, numerous amino acids and a variety of nitrostyrenes.Initially, to investigate the effect of the increment in the carbon chain of amino acids on the yield of the reaction, isatin/5-bromo isatin 1a-b and various amino acids 2a-e were allowed to react with E-1,4-dimethoxy-2-(2-nitrovinyl)benzene 3a using the optimized reaction conditions (Scheme 2).One carbon containing amino acids, i.e., in the case of alanine 2b, when reacted with isatin and 5-bromo isatin 1a-b, it furnished the products 4b and 4e in excellent (±) 91% and 85% combined yields, respectively.
one; therefore, it generated the corresponding product in relatively good yields.In addition, the secondary cyclic amino acid was efficient at generating the desired product in excellent yields compared to its counterpart (aliphatic amino acids).To conclude, we have synthesized twenty-three novel nitrostyrene-based spirooxindoles in good to excellent yields.

Single Crystal X-ray Diffraction Studies
Finally, the stereochemistry of the novel synthesized spiro[pyrrolidine-2,3 -oxindole] analogs via [1,3] dipolar cycloaddition (Huisgen) reaction was unequivocally determined by the single crystal X-ray diffraction analysis of the cycloadduct 4l (Figure 3, Page S1-S2 of SI).A suitable single crystal of compound 4l was prepared and the diffraction data were collected on a Rigaku Oxford Diffractometer.The OLEX2 [83,84] program was used to solve the structure using direct methods and refined with the ShelXL [85] refinement package using least squares minimization.The single crystal X-ray analysis confirmed all the stereochemistry associated and, overall, the three-dimensional structure of the compound 4l. Figure 3 represents an ORTEP diagram drawn with 30% probability displacement ellipsoids.The phenyl ring is found to be disordered due to the presence of two major rotamers [C19, C20, C21, C22, C23 C24 and C19, C20A, C21A, C22, C23A, C24A].Along with single crystal X-ray data of 4l, the structures of spiro[pyrrolidine-2,3 -oxindole] have been confirmed by 1 H and 13 C NMR and HRMS spectroscopic techniques.The full characterization data are reported in the Materials and Methods section; in addition, the 1 H and 13 C NMR spectral data of 4a-w are given in Supplementary Materials.Finally, the stereochemistry of the novel synthesized spiro[pyrrolidine-2,3′-oxindole] analogs via [1,3] dipolar cycloaddition (Huisgen) reaction was unequivocally determined by the single crystal X-ray diffraction analysis of the cycloadduct 4l (Figure 3, Page S1-S2 of SI).A suitable single crystal of compound 4l was prepared and the diffraction data were collected on a Rigaku Oxford Diffractometer.The OLEX2 [83,84] program was used to solve the structure using direct methods and refined with the ShelXL [85] refinement package using least squares minimization.The single crystal X-ray analysis confirmed all the stereochemistry associated and, overall, the three-dimensional structure of the compound 4l. Figure 3 represents an ORTEP diagram drawn with 30% probability displacement ellipsoids.The phenyl ring is found to be disordered due to the presence of two major rotamers [C19, C20, C21, C22, C23 C24 and C19, C20A, C21A, C22, C23A, C24A].Along with single crystal X-ray data of 4l, the structures of spiro[pyrrolidine-2,3′-oxindole] have been confirmed by 1 H and 13 C NMR and HRMS spectroscopic techniques.The full characterization data are reported in the Materials and Methods section; in addition, the 1 H and 13 C NMR spectral data of 4a-w are given in Supplementary Materials.

Plausible Mechanism
It has been well documented that the spirooxindole class of bioheterocycles possesses a wide array of biological activity.However, we realized during our investigation that nitrostyrene-based spirooxindoles have never been subjected to testing of their anticancer activity in spite of having a nitro group which is responsible for cytotoxicity [45][46][47].Therefore, in our ongoing search for a novel anticancer agent, we have synthesized 23 novel amino acid-nitrostyrene-based spirooxindoles by utilizing the literature procedure [72].Additionally, with reference to the literature [70,72], the plausible mechanism of the regio-/stereoselective product can be depicted starting from the stereochemistry of the four stereocenters that can possibly have eight pairs of promising diastereoisomers, but the predefined stereochemistry of amino acid and trans β-nitrostyrene will determine the stereochemistry of C-1, C-2 and C-3 in the product.Subsequently, the non-appearance of six diastereoisomer pairs has been anticipated due to two reasons: (i) the hydrogen at C-3 cannot have a symmetry that is above the plane; and (ii) there is no possibility of the substituents at C-1 and C-2 to attain Z-configuration.Considering the stereochemistry of the quaternary carbon, previous reports [70,72] depicted how the reaction between isatin and amino acid give rise to the formation of two possible intermediates.i.e., I2 and I2′; interestingly, out of these two, only one intermediate I2 is highly stable and observed in our case of regioselective product.The probable reason can be justified from the hydrogenbond interaction influenced by the carbonyl group present on isatin and so, we get one intermediate exclusively (Scheme 3).

Plausible Mechanism
It has been well documented that the spirooxindole class of bioheterocycles possesses a wide array of biological activity.However, we realized during our investigation that nitrostyrene-based spirooxindoles have never been subjected to testing of their anticancer activity in spite of having a nitro group which is responsible for cytotoxicity [45][46][47].Therefore, in our ongoing search for a novel anticancer agent, we have synthesized 23 novel amino acid-nitrostyrene-based spirooxindoles by utilizing the literature procedure [72].Additionally, with reference to the literature [70,72], the plausible mechanism of the regio-/stereoselective product can be depicted starting from the stereochemistry of the four stereocenters that can possibly have eight pairs of promising diastereoisomers, but the predefined stereochemistry of amino acid and trans β-nitrostyrene will determine the stereochemistry of C-1, C-2 and C-3 in the product.Subsequently, the non-appearance of six diastereoisomer pairs has been anticipated due to two reasons: (i) the hydrogen at C-3 cannot have a symmetry that is above the plane; and (ii) there is no possibility of the substituents at C-1 and C-2 to attain Z-configuration.Considering the stereochemistry of the quaternary carbon, previous reports [70,72] depicted how the reaction between isatin and amino acid give rise to the formation of two possible intermediates.i.e., I2 and I2 ; interestingly, out of these two, only one intermediate I2 is highly stable and observed in our case of regioselective product.The probable reason can be justified from the hydrogenbond interaction influenced by the carbonyl group present on isatin and so, we get one intermediate exclusively (Scheme 3).Once the stereochemistry of the quaternary stereocenter has been determined, the potential development of an extra pair of diastereoisomers is dismissed and the following errand was to decide the stereochemistry of the substituents at positions C-1′ and C-2′ of cycloadduct 4, whose stereoselectivity will be resolved by the [3+2] cycloaddition between the azomethine ylide I3 (Scheme 3) and trans β-nitrostyrene 3. The key factor here is the ylide structure because of its cyclic nature which gives a fairly inflexible ring layout and brings about a superior diastereofacial approach between the ylide and the β-nitrostyrene.Once the stereochemistry of the quaternary stereocenter has been determined, the potential development of an extra pair of diastereoisomers is dismissed and the following errand was to decide the stereochemistry of the substituents at positions C-1 and C-2 of cycloadduct 4, whose stereoselectivity will be resolved by the [3+2] cycloaddition between the azomethine ylide I3 (Scheme 3) and trans β-nitrostyrene 3. The key factor here is the ylide structure because of its cyclic nature which gives a fairly inflexible ring layout and brings about a superior diastereofacial approach between the ylide and the β-nitrostyrene.
The approach between the two can be interpreted in two different ways.We start with the exo approach which portrays that the steric hindrance between the basic isatin skeleton and cumbersome trans β-phenyl nitrostyrene brings about an electrostatic aversion within these components resulting in an increase in the free energy of activation for that particular transition state whereas the trans β-phenyl nitrostyrene will adequately escape from the isatin moiety in the endo approach and thus form a stable transition state which has a low value of free energy of activation.Therefore, cycloadduct 4 will be profoundly observed through the endo-approach pathway while the stereoisomer I3a, obtained from the exo-approach, was definitely not found in our reaction (Scheme 4).

In Vitro Anticancer Activity against Lung/Liver Cancer and Normal Cell Lines
All the synthesized derivatives of spiro[pyrrolidine-2,3′-oxindoles] 4a-w have been evaluated for their in vitro cytotoxic activities against liver and lung cancer cell lines as well as immortalized normal cell lines.While human lung cancer (A549) cell lines were compared to immortalized (BEAS-2B) normal cell lines, the liver cancer was scrutinized by using HepG2 cancer liver cell lines versus LO2 normal liver cell lines.The study used artemisinin, artesunic acid, chloroquine and paclitaxel as standard reference drugs (Tables 2 and 3, Supplementary Materials Figures S1-S4).
It has been concluded that the bromo-substituted isatin was found to be more fruitful in comparison to the unsubstituted isatin.Subsequently, amino acids such as leucine, alanine and proline were blended and helped to enhance the anticancer activity of corresponding compounds.While talking about the effects of various groups on the anticancer activity against the lung (A549) cancer cell lines, the 1,4-dimethoxynitrostyrene and (E)-2-(benzyloxy)-1-methoxy-4-(2-nitrovinyl)benzene were the only two nitroaromatic styrenes that enhanced the anticancer activity of the spirooxindole derivatives.Out of these few selective spirooxindole derivatives, compound 4c was found to be the most active of the series with the IC 50 value of 34.99 µM against lung A549 cancer cell lines with a selectivity index of 0.96 (Table 2, entry 3), whereas compounds 4f, 4m, 4q, and 4t exhibited equally comparable results to those of compound 4c and showed an IC 50 value of 41.12, 45.94, 47.92 and 45.22 µM, respectively (Table 2, entries 6,13,17,20).Although the IC 50 values of these derivatives were slightly lower, they had a better selectivity index than compound 4c, which is 2.43, 1.38, 2.09 and 1.50, respectively.The derivative 4e was the least active out of these six having an IC 50 value of 85.63 µM and a selectivity index of 1.17 (Table 2, entry 5).Moreover, these compounds were found to be more active than artemisinin and chloroquine.They were quite potent and comparable with artesunate and showed these promising activities.However, all the compounds were found to have a less promising activity profile compared to the standard drug paclitaxel.
In terms of the factors affecting the cytotoxicity of the prepared substrates, it almost follows a similar pattern as that against the lung cancer cell lines but the values of minimum inhibitory concentration (MIC) come out to be slightly higher than the previous cancer cell lines.Out of these six derivatives, compound 4m exhibited the most promising anticancer activity.Unlike the lung cancer cell lines, six derivatives showed higher IC 50 values against liver cancer cells but also have relatively lower selectivity index (SI) values.This signifies that the prepared spirooxindole derivatives were more efficient against lung cancer cell lines than liver cancer cell lines.

General Information
Oven-dried laboratory glassware was used for all the synthetic procedures.Melting points were taken in open capillaries on a Sisco melting point apparatus and are presented uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a JEOL ECS-400 spectrometer (2-channel console with a flexible broadband RF performance), which was operating at 400 MHz for 1 H and 100 MHz for 13 C NMR and utilized CDCl 3 as a solvent for all the sample preparations.Tetramethylsilane (δ 0.00 ppm) and CDCl 3 both were served as an internal standard in 1 H NMR (δ 7.246 ppm) and 13 C (δ 77.0 ppm) NMR.Patterns of chemical shifts were reported in parts per million.Peak splitting patterns were described as singlet (s), broad singlet (brs), doublet (d), double doublet (dd), triplet (t) and multiplet (m).Coupling constants (J) were reported in Hertz (Hz).High-Resolution Electron Impact Mass Spectra (HR-EIMS) were obtained on Xevo G2-S Q-Tof (Waters, Milford, MA, USA) compatible with ACQUITY UPLC ® and nano ACQUITY UPLC ® systems.Column chromatography was performed over normal (particle size: 60-120 Mesh, 100-200 Mesh) and flash (particle size: 230-400 Mesh) silica gel, which were procured from QualigensTM (Mumbai, India), Rankem (Haryana, India), and Spectrochem (Mumbai, India).TLC plates coated with silica gel (Kiesel 60-F254, Merck (Bengaluru, India) were used for the monitoring of the progress of the reactions.Visualizing agents used for TLC were UV light.BUCHI's Rotavapor R-210 was used for all drying and concentration procedures.All the analytical-grade supplied solvents such as MeOH and EtOH were used without further purification.All the remaining chemicals and reagents obtained from Sigma Aldrich (Bangalore, India), or Merck (Bengaluru, India) and TCI (Chennai, India) were used without further purification.

General Procedure for the Synthesis of (E)-2-Aryl-1-nitroethenes (3a-e)
The substituted benzaldehyde (1.0 mmol) and nitromethane (5.0 mmol) were added dissolved in the glacial acetic acid.The ammonium acetate (2.5 mmol) was added into the reaction mixture and the whole reaction mixture was sonicated at 60 • C for 4-5 h (Scheme 5) [86].After the completion of the reaction mixture, the reaction mixture was concentrated under reduced pressure using rotavapor to get the crude mixture, which was further purified through silica-gel column chromatography using the ethyl acetate/hexane (03:97 as an eluent to furnish the pure product 3a-e in 82-85% yield as a yellow solid. The substituted benzaldehyde (1.0 mmol) and nitromethane (5.0 mmol) were added dissolved in the glacial acetic acid.The ammonium acetate (2.5 mmol) was added into the reaction mixture and the whole reaction mixture was sonicated at 60 °C for 4-5 h (Scheme 5) [86].After the completion of the reaction mixture, the reaction mixture was concentrated under reduced pressure using rotavapor to get the crude mixture, which was further purified through silica-gel column chromatography using the ethyl acetate/hexane (03:97 as an eluent to furnish the pure product 3a-e in 82-85% yield as a yellow solid.Scheme 5. Synthesis of (E)-2-aryl-1-nitroethenes (3a-e).

Scheme 2 .
Scheme 2. a Reaction Conditions: Substituted isatins 1a-b (0.5 mmol), various amino acids (0.5 mmol and nitrostyrenes 3a-e dissolved in methanol as solvent at 60 °C for 15 min under microwave irradiation.b Combined yield of isolated mixture of regioisomers.
[80] 50 : Concentration at which the inhibition of 50% cells was observed.Values expressed are means from at least three independent experiments conducted in duplicates; b S.I.: Selectivity index lung (IC 50 for cytotoxicity against normal cells/IC 50 for cytotoxicity against cancer cells); c See ref.[80].